Lymphocytes in the development of lung inflammation: a role for regulatory CD4+ T cells in indirect pulmonary lung injury

Abstract

Although roles for myelocytes have been suggested in the pathophysiology of indirect acute lung injury (ALI not due to a direct insult to the lung), the contribution of various regulatory lymphoid subsets is unknown. We hypothesized a role for lymphocytes in this process. Using a sequential model of indirect ALI induced in mice by hemorrhagic shock followed 24 h later by polymicrobial sepsis; we observed a specific and nonredundant role for each lymphocyte subpopulation in indirect ALI pathophysiology. In particular, we showed that CD4(+) T cells are specifically recruited to the lung in a dendritic cell-independent but IL-16-dependent process and diminish neutrophil recruitment through increased IL-10 production. Most importantly, this appears to be mediated by the specific subpopulation of CD4(+)CD25(+)Foxp3(+) regulatory T cells. Although indirect ALI has constantly been described as a proinflammatory pathology mediated by cells of the innate immune system, we now demonstrate that cells of the adaptive immune response play a major role in its pathophysiology as well. Most importantly, we also describe for the first time the nature of the regulatory mechanisms activated in the lung during indirect ALI, with CD4(+) regulatory T cells being central to the control of neutrophil recruitment via increased IL-10 production.

FIGURE 1

T lymphocytes are recruited to the lung during indirect ALI in mice. Indirect ALI was induced in mice by Hem followed 24 h later by a polymicrobial septic challenge (CLP). As controls, mice were submitted to sham Hem + CLP or sham Hem + sham CLP. Lungs were harvested 24 h after CLP, enzymatically digested, and flow cytometry was performed. A, Total number of lymphocytes (Ly) per ml was calculated based on cell count and percentage of lymphocytes determined on a CD45/side scatter flow cytometry dot plot. Results are expressed as number of 10⁵ lymphocytes per ml of lung digest. B, The respective percentages of T, B, NK, and NK T cells were then measured by flow cytometry and total numbers were calculated. Results are expressed as number of 10⁵ lymphocytes per ml of lung digest. C and D, The percentages of γδ T cells and of CD4⁺ and CD8⁺ lymphocytes were then measured and absolute numbers were calculated. C, Results are expressed as number of 10⁴ γδ T lymphocytes per ml of lung digest and D, as ratio between numbers of CD4⁺ and CD8⁺ T cells. Values are expressed as mean ± SEM (n = 7–9 mice/group). #, p < 0.05 vs sham Hem-sham CLP.*, p < 0.05 vs sham Hem + CLP (Mann-Whitney U test).

FIGURE 2

T cell recruitment to the lung during indirect ALI is independent of the presence of DCs but depends on lung IL-16. A and B, CD11c-DTR and C57BL/6J (CTRL) mice were injected with diphtheria toxin (4 μg/kg) 12 h before the induction of indirect ALI by Hem followed 24 h later by a polymicrobial septic challenge (CLP). As controls, mice were submitted to sham Hem + CLP. Lungs were harvested 24 h after CLP, enzymatically digested, and flow cytometry was performed (n = 7–9 mice/group). A, The percentage of lung CD11c⁺MHC II⁺ cells was measured and absolute numbers were calculated. Results are presented as number of 10⁴ CD11c⁺MHC II⁺ cells per ml of lung digest. B, The percentage of CD3⁺ T cells was monitored by flow cytometry and absolute numbers were calculated. Results are expressed as number of 10⁵ T lymphocytes (Ly) per ml of lung digest. C, C57BL/6J mice were submitted to Hem followed 24 h later by CLP. As controls, mice were submitted to sham Hem + CLP or sham Hem + sham CLP. Lungs were harvested and IL-16 levels were measured by ELISA in lung homogenates. Results are expressed as pg/mg of IL-16 in lung homogenate (n = 4 mice/group). D, C57BL/6J mice were injected IT with anti-IL-16-blocking Abs (anti-IL16 Abs) or IgG (75 μg/mouse) 12 h before the induction of indirect ALI by Hem followed 24 h later by CLP. Lungs were harvested 24 h after CLP, enzymatically digested, and flow cytometry was performed. The percentage of lung CD3⁺CD4⁺ cells was measured and absolute numbers were calculated. Results are presented as number of 10⁴ CD4⁺ T cells per ml of lung digest (n = 4–7 mice/group). E, Lung ECs were purified from C57BL/6J mice after Hem + CLP or CLP alone. mRNA expression for IL-16 was measured by densitometric evaluation of PCR results. β -Actin served as internal control (n = 3 mice/group). Values are expressed as mean ± SEM. #, p < 0.05 vs respective sham Hem-sham CLP. *, p < 0.05 vs respective sham Hem-CLP. @, p < 0.05 vs control or IgG Hem + CLP group (Mann-Whitney U test). Ly, Lymphocyte.

FIGURE 3

RAG-deficient mice present with increased lung dysfunctions during indirect ALI. Indirect ALI was induced in C57BL/6J mice (CTRL) and RAG-deficient mice by Hem followed 24 h later by a polymicrobial septic challenge (CLP). As controls, mice were submitted to sham Hem + CLP. A, Protein concentration, as a maker for lung leakage, was measured in the BALFs. B, Lungs were fixed, sectioned, and stained with H&E. Representative sections are shown for each mouse group (original magnification, ×200). C, Lung apoptosis was monitored in whole lung homogenate by the measurement of caspase 3 activity. D, Neutrophil recruitment was monitored by the measurement of MPO activity in lung homogenate. E, Cytokine expressions were measured in lung homogenate by ELISA. Values are expressed as mean ± SEM (n = 7–9 mice/group). @, p < 0.05 vs control Hem + CLP group.*, p < 0.05 vs respective sham Hem-CLP group (Mann-Whitney U test).

FIGURE 4

Specific and nonredundant roles of lymphocyte subpopulations in the pathophysiology of indirect ALI. CD8⁺ T cell (CD8^−/−)-, γδ T cell (γδ^−/−)-, CD4⁺ T cell (CD4^−/−)-, B cell (B^−/−)-deficient and C57BL/6J (CTRL) mice were submitted to Hem followed 24 h later by a polymicrobial septic challenge (CLP) to induce indirect ALI. As controls, mice were submitted to sham Hem (Sh) + CLP. A, Lung apoptosis was monitored in whole lung homogenate by the measurement of caspase 3 activity. B, Neutrophil recruitment was monitored by the measurement of MPO activity in lung homogenate. C, IL-10 level was measured in lung homogenate by ELISA. D, Control mice were submitted to IT instillation of anti-CD4-depleting Abs or IgG (50 μg/mice) 12 h before Hem. Anti-CD4 Abs led to the significant down-regulation of lung CD4⁺ lymphocyte percentage as monitored by flow cytometry (data not shown). Lungs were fixed, sectioned, and stained with H&E. Representative sections are shown for each mouse group (original magnification, ×200). Values are expressed as mean ± SEM (n = 5–9 mice/group). @, p < 0.05 vs control Hem + CLP group.*, p < 0.05 vs respective sham Hem-CLP group (Mann-Whitney U test).

FIGURE 5

IL-10 blockade recapitulates the observations made in CD4-deficient/depleted mice after Hem + CLP. C57BL/6J mice received an i.p. injection of anti-mouse IL-10R-neutralizing Ab (anti-IL10R Abs) or IgG (250 μg/mice) 12 h before the induction of indirect ALI by Hem followed 24 h later by polymicrobial septic challenge (CLP). As controls, mice were submitted to sham Hem + CLP. A, Protein concentration, as a maker for lung leakage, was measured in the BALFs. B, Lungs were fixed, sectioned, and stained with H&E. Representative sections are shown for each group (original magnification, ×200). C, Lungs were digested and cells were phenotyped for GR1 expression as a marker for neutrophils. Absolute counts were then calculated. Percentage of GR1⁺ cells among total leukocytes (CD45⁺ cells) and number of GR1⁺ cells (10⁵) per ml of lung digest are shown. D, Neutrophil recruitment was monitored in lung homogenate by the measurement of MPO activity. E, KC levels were measured in lung homogenate by ELISA. Values are expressed as mean ± SEM (n = 4 mice/group). Dashed line represents sham Hem + CLP mice. @, p < 0.05 vs IgG-treated Hem + CLP group. *, p < 0.05 vs sham Hem-CLP group (Mann-Whitney U test).

FIGURE 6

Decreasing Foxp3 expression in the lung recapitulates the effects observed in CD4-deficient/depleted mice and in anti-IL10R Ab-treated mice after Hem + CLP. A–D, EGFP-Foxp3 mice were submitted to IT instillation of cationic liposome (DOTAP)- encapsulated Foxp3-targeting (Foxp3) or nontargeting control siRNA (CTRL) 12 h before the induction of indirect ALI by Hem followed 24 h later by polymicrobial septic challenge (CLP). As controls, mice were submitted to sham Hem + CLP. A, Lungs were digested and cells were phenotyped for Foxp3, CD3, CD4, and CD25 expressions. Representative dot plots are shown for coexpression of CD25 and Foxp3. Percentages in the top right quadrants refer to percentages of cells positive for the four markers measured among total lymphocytes selected based on a CD45/side scatter dot plot. B, The percentage of Foxp3⁺CD4⁺CD25⁺ T cells among total lymphocytes was measured by flow cytometry and the absolute count was calculated. Results are expressed as percentages of Foxp3⁺CD4⁺CD25⁺CD3⁺ cells among total lymphocytes and as number of Foxp3⁺CD4⁺CD25⁺ T cells (10⁴) per ml of lung digest.. C, Lungs were fixed, sectioned, and stained with H&E. Representative sections are shown for each group (original magnification, ×200). D, The IL-10 level was measured in lung homogenate by ELISA. E, Lung CD4⁺CD25⁺ cells were purified from C57BL/6J mice after Hem + CLP or CLP alone. mRNA expression for IL-10 and TGF-β was measured by densitometric evaluation of PCR results. β -Actin served as internal control. Values are expressed as mean ± SEM (n = 4 mice/group). Dashed line represents sham Hem + CLP mice. @, p < 0.05 vs control Hem + CLP group.*, p < 0.05 vs sham Hem-CLP group (Mann-Whitney U test).